ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer mortality in the US, with 5- year survival rates between 0.4 and 4%. Although development of more effective therapies remains an important, unmet need, two effective chemotherapy regimens have recently been developed. Clinicians remain without tools to choose between these two regimens when treating patients in the frontline setting, or to choose chemotherapy regimens in the second line setting or later. Furthermore, a greater understanding regarding the key genes and pathways responsible for treatment resistance and disease biology is required. We have conducted and published promising results of a clinical trial, a highly collaborative effort between the academic and industrial PIs, demonstrating that a simple blood test is capable of predicting effective chemotherapy treatment for individual patients with advanced PDAC. In a separate, recently published study, we have developed an innovative and powerful organoid culture system for studying human PDAC. We propose to expand upon these findings by conducting translational clinical trials focused on patients with advanced PDAC. The blood test already developed will be optimized and validated. Equally important, the organoid model will be leveraged to study the genomic basis for treatment resistance and metastasis. Furthermore, an invaluable organoid resource will be established for future studies. We will test the hypotheses that: 1) optimizing and applying our simple blood test to actively guide treatment in advanced PDAC will improve patient survival, and 2) further characterization of CTICs will lead to biological and therapeutic insights. We propose to test these hypotheses under the following three highly integrated Specific Aims: Aim 1: Validate and optimize our existing blood test for predicting optimal therapy. A translational, clinical trial will be conducted to optimize a PGx assay for guiding chemotherapy treatment of advanced PDAC. PGx profiles from CTCs isolated using two different and innovative approaches (selection based on invasive phenotype or size) and from tumor tissue biopsies will be assessed. Aim 2: Employ our innovative organoid approach for PGx profiling and to understand CTC and tumor derived organoids. Newly developed organoid methodology will be used to expand rare and heterogeneous circulating and tumor-derived cells, collected both at baseline and at disease progression. Performance of organoid PGx profiling will be compared to that of CTICs. CTC and organoid genetics and biology will be explored. Aim 3: Validate an optimized clinical assay to guide chemotherapy treatment of advanced PDAC. A pivotal, prospective clinical trial will be conducted. Beyond choosing from standard therapies in the frontline, novel/targeted therapies will be explored in subsequent lines. An invaluable, well-annotated human organoid repository will be established to accelerate study of disease biology and therapeutics. These aims will be completed as part of a highly collaborative effort between the academic and industry PIs. We believe that the successful completion of the proposed, innovative research will result in a clinically useful test to improve survival for patients with advanced PDAC, insights into disease biology, and creation of a biorepository resource for biological and therapeutic study.